Image from Tyler et al. 2012, MPMI. http://dx.doi.org/10.1094/MPMI-02-13-0051-IA. Fig. 2. Specific microbe-independent entry of Avr1bNt proteins into soybean root cells and wheat leaf cells. A-E, Soybean root entry by Avr1b N-terminus (residues 1-50) labeled by Dylight488, and counter stained with propidium iodide and 4',6-diamidino-2-phenylindole (DAPI). F-J, Soybean root entry by Avr1b N-terminus (residues 1-50) labeled by Dylight488, mixed with Avr1b N-terminus with RFLR->qFLR mutation labeled with DyLight550, and counter stained with DAPI. A,F, light image; B,G, Dylight488 image; C, propidium image; H, Dylight550 image; D,I,DAPI image; E,J, overlay of the three fluorescent images. In A-J, purified fusion proteins (0.4 mg/ml each) were incubated with soybean (Williams) root tips, for 2 hours at 25oC in PBS buffer adjusted to pH 7.2, then washed for 15 min in formalin (PBS + 10% formaldehyde) containing 0.2 g/ml propidium iodide (A-E only) and 0.4 g/ml DAPI. K-P, Wheat leaf cell entry by Avr1b N-terminus (residues 1-50) labeled by Dylight488 mixed with Avr1b N-terminus (residues 1-50) with RFLR->qFLR mutation labeled by Dylight550. Purified fusion proteins (0.4 mg/ml each) in PBS buffer adjusted to pH 7.2 were infiltrated with a blunt syringe into ~9 day old wheat seedling leaves. Leaves were imaged after 6 h without washing. K, chloroplast fluorescence (excitation 488 nm; emission meta filter 675-715 nm); L, Dylight550 image (excitation 543 nm; emission window 585-615 nm; gain 600 to 654; digital offset -0.1 to 0); M, Dylight488 image (excitation 488 nm; emission window 505-530 nm; gain 580 to 610; digital offset -0.1 to 0); N, overlay of K-M; O, light image; overlay of N and O. Labeling of proteins with Dylight dyes was as described (Sun et al., 2013). The experiments shown in this figure were conducted at Virginia Tech using a Zeiss LSM 510 Meta confocal microscope.